Alpha-Mannosidosis
Alpha-mannosidosis is a recessive, autosomal disease that occurs world wide with a frequency of between 1/1,000,000 and 1/500,000. Mannosidosis is found in all ethnic groups in Europe, America, Africa and also Asia. It is detected in all countries with a good diagnostic service for lysosomal storage disorders, at a similar frequency. They are born apparently healthy, however the symptoms of the diseases are progressive. Alpha-mannosidosis displays clinical heterogeneity, ranging from very serious to very mild forms. Typical clinical symptoms are: mental retardation, skeletal changes, impaired immune system resulting in recurrent infections, hearing impairment and often the disease is associated with a typical facial characteristics such as a coarse face, a prominent forehead, a flattened nasal bridge, a small nose, and a broad mouth. In the most severe cases (mannosidosis type I) the children suffer from hepatosplenomegaly, and they die during the first years of life. Possibly this early death is caused by severe infections due to the immunodeficiency caused by the disease. In milder cases (mannosidosis type 2) the patients usually reach adult age. The skeletal weaknesses of the patients result in the needs of wheeling chairs at age 20 to 40. The disease causes a diffuse dysfunction of the brain often resulting in weak mental performances that excludes anything but the most basic skills of simple reading and writing. These problems associated with hearing inabilities and other clinical manifestations preclude the patient from an independent life, the consequence being that life long caretaking is needed.
Lysosomal Alpha-Mannosidase
Alpha-mannosidosis results from a deficient activity of lysosomal alpha-mannosidase (LAMAN, EC3.2.1.24). The disease is characterised by massive intracellular accumulation of mannose-rich oligosaccharides, that is oligosaccharides carrying α1,2-, α1,3- and α1,6-mannosyl residues at their non-reducing termini. These oligosaccharides mainly originate from the intralysosomal degradation of glycoproteins with N-linked oligosaccharides. However, some originate from the catabolism of dolichol-linked oligosaccharides and from misfolded glycoproteins redirected to the cytosol for degradation by the proteasome (Hirsch et al. EMBO J. 22, 1036-1046, 2003 and Saint-Pol et al. J. Biol. Chem. 274, 13547-13555, 1999). The lysosomal storage is observed in a wide range of cell types and tissues, including neurons in all brain regions. LAMAN is an exoglycosidase which hydrolyses these terminal, non-reducing alpha-D-mannose residues in alpha-D-mannosides from the non-reducing end during the ordered degradation of the N-linked glycoproteins (Aronson and Kuranda FASEB J 3:2615-2622. 1989). The human precursor enzyme is synthesised as a single polypeptide of 1011 amino acids including a signal peptide of 49 residues. The precursor is proteolytically processed into three main glycopeptides of 15, 42, and 70 kD to the matured enzyme in the lysosome. The 70 kD glycopeptide is further processed into three subunits linked by disulfide bridges. (Berg et al. Mol. Gen. and Metabolism 73, 18-29, 2001, Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997).
The Lysosomal Alpha-Mannosidase Gene
The gene coding for LAMAN (MANB) is located at chromosome 19 (19cen-q12), (Kaneda et al. Chromosoma 95:8-12. 1987). MANB consists of 24 exons, spanning 21.5 kb (GenBank accession numbers U60885-U60899; Riise et al. Genomics 42:200-207, 1997). The LAMAN transcript is >>3,500 nucleotides (nts) and contains an open reading frame encoding 1,011 amino acids (GenBank U60266.1).
The cloning and sequencing of the human cDNA encoding LAMAN has been published in three papers (Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997; Liao et al. J. Biol. Chem. 271, 28348-28358. 1996; Nebes et al. Biochem. Biophys. Res. Commun. 200, 239-245. 1994). Curiously, the three sequences are not identical. When compared to the sequence of Nilssen et al (accession # U60266.1) a TA to AT change at positions 1670 and 1671 resulting in a valine to asparitic acid substitution was found by Liao et al. and Nebes et al. Also a C to A change in pos 1152 was found which do not result in any changes in the amino acid sequence.
Diagnosis
The diagnosis of alpha-mannosidosis is currently is based on clinical evaluation, detection of mannose-rich oligosaccharides in urine, and direct measurements of alpha-mannosidase activity in various cell types, such as leukocytes, fibroblasts, and amniocytes (Chester et al., In: Durand P, O'Brian J (eds) Genetic errors of glycoprotein metabolism. Edi-Ermes, Milan, pp 89-120. 1982; Thomas and Beaudet. In: Scriver C R, Beaudet A L, Sly W A, Valle D (eds) The metabolic and molecular bases of inherited disease. Vol 5. McGraw-Hill, New York, pp 2529-2562. 1995).
Because the symptoms initially often are mild and the biochemical diagnosis is difficult, the diagnosis is frequently made late in the course of the disease. It is obvious that patients and their families would benefit substantially from an early diagnosis.
Animal Models
Alpha mannosidosis has been described in cattle (Hocking et al. Biochem J 128:69-78. 1972), cats (Walkley et al. Proc. Nat. Acad. Sci. 91: 2970-2974, 1994), and guinea pigs (Crawley et al. Pediatr Res 46: 501-509, 1999). A mouse model was recently generated by targeted disruption of the alpha-mannosidase gene (Stinchi et al. Hum Mol Genet 8: 1366-72, 1999).
Like in humans alpha mannosidase seems to be caused by specific mutations in the gene coding for lysosomal alpha-mannosidase. Berg et al. (Biochem J. 328:863-870.1997) reported the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consists of 3 polypeptides, with molecular masses reported to be 72, 41, and 12 kD. Similary to the human enzyme it was demonstrated that the feline enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the 3 polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1% and 83.2% identical with the human and bovine sequences, respectively. A 4-bp deletion was identified in an affected Persian cat; the deletion resulted in a frameshift from codon 583 and premature termination at codon 645. No enzyme activity could be detected in the liver of the cat. A domestic long-haired cat expressing a milder phenotype had enzyme activity of 2% of normal; this cat did not possess the 4-bp deletion. Tollersrud et al. (Eur J Biochem 246:410-419. 1997) purified the bovine kidney enzyme to homogeneity and cloned the gene. The gene was organized in 24 exons that spanned 16 kb. Based on the gene sequence they identified two mutations in cattle.
Medical Need for Alpha-Mannosidosis Therapy
In light of the severe clinical manifestations resulting from the accumulation of mannose-rich oligosaccharides, the lack of effective treatment for alpha-mannosidosis is well recognised. At present, the major therapeutic option for treatment of the disease is bone marrow transplantation, however, it is the aim of the present invention to promote enzyme replacement therapy as a potential future alternative.
Bone Marrow Transplantation
In 1996 Walkley et al. (Proc. Nat. Acad. Sci. 91: 2970-2974, 1994) published a paper on 3 kittens with mannosisdosis that were treated with bone marrow transplantation (BMT) in 1991. In the 2 animals that were sacrificed a normalisation was seen, not only in the body, but more importantly, also in brain. The 3'rd cat was well after 6 years. Normally, an untreated cat dies with 3-6 months. In 1987 a child with mannosidosis was treated with BMT (Will et al. Arch Dis Child 1987 October; 62(10):1044-9). He died after 18 weeks due to procedure related complications. In brain little enzyme activity was found. This disappointing result could be explained by heavy immunosuppressive treatment before death, or that it takes time for the enzyme activity to increase in brain after BMT. The donor was the mother (who as carrier must be expected to have less than 50% enzyme activity) or it may be BMT in man has no effect on enzyme function in brain. Despite having variable outcomes the few attempts of bone marrow transplantation have thus indicated that successful engraftment can correct the clinical manifestations of alpha-mannosidosis, at least in part. However, the challenge of reducing the serious procedure related complications when applying bone marrow transplantation in human therapy still remains to be defeated.
Enzyme Replacement Therapy
When lysosomal storage diseases were discovered, hopes were raised that this could be treated by enzyme substitution. Enzyme replacement therapy has proven efficient in Gaucher disease. When exogenous lysosomal glucocerebrosidase is injected into the patient, this enzyme is taken up by enzyme-deficient cells (Barton et al. N Engl J Med 324:1464-1470). Such uptake is regulated by certain receptors on the cell surface as for instance the mannose-6-phosphate receptor, which is nearly ubiquitous on the surface of cells and other receptors such as the asialoglycoprotein receptor and the mannose receptor, which are restricted to certain cell types such as cells of the monocyte/macrophage cell line and hepatocytes. The cellular uptake of the enzyme is therefore heavily dependent upon its glycosylation profile. If properly designed, the deficient enzyme could be replaced by regular injections of exogenous enzyme in the same manner as diabetic patients receive insulin. In vitro studies with the purified active lysosomal alpha-mannosidase added to the media of enzyme-deficient fibroblasts showed correction of the lysosomal substrate accumulation. In vivo treatment, on the other hand, has been hampered in part by the problem of producing the sufficient quantity of enzymes, due to difficult large scale production and purification procedures, and by complications resulting from immune reactions against the exogenous enzyme.
Most importantly, however, special considerations apply in relation to lysosomal storage diseases with a major neurological component, such as alpha-mannosidosis, wherein the clinical manifestations are related to increased lysosomal storage within the central nervous system. Thus, enzyme replacement therapy has not proven effective against the acute neuronopathic variant of Gaucher disease (Prows et al. Am J Med Genet 71:16-21).
The delivery of therapeutic enzymes to the brain is prevented by the absence of transport of these large molecules through the blood-brain barrier. From the general notion that the blood brain barrier must be circumvented in order to see an effect of therapeutic agents in the brain, the use of a large diversity of delivery systems have been contemplated. These include invasive techniques such as osmotic opening of the blood brain barrier with for instance mannitol and non-invasive techniques such as receptor mediated endocytosis of chimeric enzymes. As enzyme replacement is expected to require administration of the enzyme on a regular basis, the use of invasive techniques should be avoided. Use of the non-invasive techniques, has only recently provided promising results in animal models (for alpha-mannosidosis see below, for other lysosomal disorders see for example: Grubb et al. PNAS 2008, 105(7) pp. 2616-2621). It has been contemplated that reduced storage in visceral organs and in the meninges could reduce the amount of oligosaccharides that is carried to the brain. Such considerations, however, are not considered to be applicable to lysosomal disorders in which the neurological damage is primary and severe (Neufeld, E. F. Enzyme replacement therapy, in “Lysosomal disorders of the brain” (Platt, F. M. Walkley, S. V: eds Oxford University Press).
However, as described in Roces et al. Human Molecular Genetics 2004, 13(18) pp. 1979-1988, Blanz et al. Human Molecular Genetics 2008, 17(22) pp. 3437-3445 and WO 05/094874 it has proven possible to increase levels of LAMAN in the central nervous system of animals using e.g. intravenous injection of a formulation comprising alpha-mannosidase thereby reducing intracellular levels of neutral mannose-rich oligosaccharides within one or more regions of the central nervous system. This indicates that recombinant alpha-mannosidase is useful in enzyme replacement therapy of patients suffering from alpha-mannosidosis. Thus, one major remaining hurdle towards providing efficient treatment of alpha-mannosidosis using enzyme replacement is providing sufficient amounts of pure recombinant alpha-mannosidase in a cost-efficient manner.
Production and Purification of Alpha-Mannosidase
WO 02/099092 discloses a small scale production process for rhLAMAN in CHO cells using serum free medium at 37° C. A small scale purification process is also described involving diafiltration of the crude enzyme and weak anion exchange chromatography using DEAE sepharose FF columns in the capture step, followed by a number of chromatographic purification steps involving hydrophobic interaction- and mixed mode chromatography.
WO 05/094874 discloses a small scale production process for rhLAMAN in Chinese Hamster Ovary (CHO) cells using serum free medium at 37° C. A small scale purification process analogous to the one of WO 02/099092 is also described.
WO 05/077093 describes the manufacture of highly phosphorylated lysozymal enzymes. In example IV a purification method for acid alpha-glucosidase (GAA) using a multi-modal resin (blue-sepharose) is described. GAA, although a lysozymal enzyme, is however entirely different from rhLAMAN. GAA is highly phosphorylated, while rhLAMAN has a low degree of phosphorylation. Furthermore, the sequence identity score is less than 12% between GAA and rhLAMAN, and finally their theoretical isoelectric points differ by more than one pH unit (5.42 and 6.48 respectively). Thus the method as described in WO 05/077093 to purify GAA is not applicable to rhLAMAN.
A small scale production process for rhLAMAN in CHO cells using 0.25% (V/V) serum and DMSO addition has been disclosed (Berg et al. Molecular Genetics and Metabolism, 73, pp 18-29, 2001. It also describes two purification processes involving a) a three-step procedure involving ultrafiltration, anion exchange chromatography and gel filtration or b) single-step immuno-affinity chromatography. It is further disclosed how method a) results in the 130 kDa enzyme fragmenting entirely into 55 kDa and 72 kDa fragments, whereas method b) results in partial fragmentation of the 130 kDa precursor into significant amounts of the 55 and 72 kDa fragments.
Hence, an improved process for production and purification of recombinant alpha-mannosidase would be advantageous. In particular, an improved process for large scale cultivation of a cell line capable of expressing alpha-mannosidase and a more efficient large scale purification process for isolating pure alpha-mannosidase with a high enzymatic activity from a cell culture would be advantageous.